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Semiconductor quantum dots (QDs) are bright fluorescence emitters with high quantum yields, high molar extinction coefficients, size-dependent tunable emission, and high photostability. [1][2][3][4][5][6] These attractive fluorescence properties prompt a wide interest in developing QD-based sensors for biological detection and imaging. [7][8][9][10][11][12][13] One often-used strategy towards the development of QD nanosensors is based on fluorescence resonance energy transfer (FRET) with the QDs as the FRET donor. There are numerous examples of FRETbased QD biosensors that include self-assembled nanocomplexes for detecting maltose, pH, 2,4,6-trinitrotoluene, thrombin, and enzyme activity. [7][8][9][10][11][12][13] In these FRET-based QD nanosensors, multiple copies of the FRET acceptor were often present on one QD, which may result in self-quenching and lead to low emission from the FRET acceptor.

This communication describes a novel protein labeling method that uses a single amino-acid tag -N-terminal cysteine residue -and small-molecule probes carrying the cyanobenzothiazole unit for specific labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity in physiological conditions, and should offer an important alternative to currently available protein labeling methods. Graphical abstract KeywordsProtein Labeling; Condensation; Terminal Cysteine; Chemical ligation; Live-cell Imaging Site-specific labeling of proteins with molecular tags enables direct visualization of protein dynamics, localization and interactions in single living cells and is a powerful tool for studying structure and function of proteins. [1] Proteins of interest can be labeled by genetic fusions to fluorescent proteins, or chemical reactions with fluorescent dyes. Chemical labeling often employs a receptor protein, for example, a mutant of human O 6 -alkylguanine-* Fax: (+1) 650-736-7925, ; Email: jrao@stanford.edu, Homepage: http://raolab.stanford.edu Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript DNA transferase, [2a] and E. coli dihydrofolate reductase, [2b] that binds to or reacts with its fluorescently tagged ligand. [2] Alternatively, smaller tags such as short peptides can be labeled by selective binding to fluorogenic dyes [3] or by enzyme-catalyzed ligation to fluorescent probes. [4] Water-compatible chemical reactions can also be applied to protein labeling, such as the Staudinger reaction between the azides and triphenylphosphines, [5] the Huisgen cycloaddition or "Click chemistry" between the azides and alkynes, [6] the reaction between aldehydes (or ketones) and aminooxy containing reagents (or hydrazides). [7] Herein, we describe a water-compatible condensation reaction for labeling terminal cysteine residues on proteins in vitro and at the cell surface.N-terminal cysteine has been frequently used in protein engineering for site-specific labeling and modification. [8] Thioesters are commonly used in a ligation reaction with terminal cysteines, which proceeds through thioester and S-to N-acyl exchanges. [9] This native chemical ligation reaction has been successfully applied to protein semi-synthesis and labeling. [10] Our method to label the terminal cysteine on a protein is based on the condensation of 2-cyanobenzothiazole (CBT) and D-cysteine, a reaction used at the last step of the synthesis of D-luciferin a common substrate for firefly luciferase (reaction 1 in Scheme 1). [11] This reaction can proceed smoothly in aqueous solutions. We hypothesized that CBT could react with the terminal cysteine on a protein. If a fluorophore is conjugated to the CBT motif, this reaction should ligate a fluorescent label specifically to the terminal cysteine of the protein (reaction...

We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few ng/ml, and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown.

Lebendig markiert: Eine Proteinmarkierungsmethode, die einen einzelnen Aminosäurelinker in Form eines N‐terminalen Cysteinrests sowie niedermolekulare Sonden mit einer Cyanbenzothiazol(CBT)‐Einheit verwendet, wurde für die spezifische Fluoreszenzmarkierung von Proteinen in vitro und auf der Oberfläche lebender Zellen eingesetzt (siehe Schema). Diese einfache Ligationsreaktion verläuft hoch spezifisch unter physiologischen Bedingungen. Rd: ein Rhodamin‐Farbstoff; TEV: Tabakmosaikvirus.

Bioluminescence resonance energy transfer (BRET) operates with biochemical energy generated by bioluminescent proteins to excite fluorophores and offers additional advantages over fluorescence energy transfer (FRET) for in vivo imaging and biosensing. While fluorescent proteins are frequently used as BRET acceptors, both small molecule dyes and nanoparticles can also serve as acceptor fluorophores. Semiconductor fluorescent nanocrystals or quantum dots (QDs) are particularly wellsuited for use as BRET acceptors due to their high quantum yields, large Stokes shifts and long wavelength emission. This review examines the potential of QDs for BRET-based bioassays and imaging, and highlights examples of QD BRET for biosensing and imaging applications. Future development of new BRET acceptors should further expand the multiplexing capability of BRET and improve its applicability and sensitivity for in vivo imaging applications.

This communication reports the use of click chemistry to site-specifically conjugate bioluminescent Renilla luciferase proteins to gold nanoparticles (Au NPs) for sensing protease activity. The bioluminescent emission from luciferase was efficiently quenched by Au NPs, but significantly recovered after the proteolytic cleavage.

GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction. Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism. Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage. Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A.